The present invention relates to compositions and methods for the treatment of various neurological diseases and neurodegenerative disorders, particularly those affected by an overabundance of Amyloid Precursor Protein (APP). In particular, it has been discovered that APP synthesis is stimulated by activation of cell surface receptors coupled to the formation of cyclic adenosine monophosphate (cAMP). Moreover, it has been found that certain substances can inhibit APP synthesis, either directly or by antagonizing receptors coupled to cAMP formation.
Alzheimer""s Disease (AD) is the most common neurodegenerative disorder of aging, and is characterized by progressive dementia and personality dysfunction. The abnormal accumulation of amyloid plaques in the vicinity of degenerating neurons and reactive astrocytes is a pathological characteristic of AD.
As the fourth leading cause of death in industrialized societies, surpassed only by heart disease, stroke and cancer, AD affects 5-11% of the population over the age of 65 and 30% of those over the age of 85. The estimated cost of caring for the approximate 2.5-4.0 million AD cases in the USA exceeded $60 billion in 1991 alone. Considering the estimated 17-25 million existing AD cases worldwide, AD will no doubt become an escalating healthcare problem of unparalleled proportions as the world""s geriatric population grows. Much work remains in the quest to find an effective treatment for AD.
APP processing is regulated by neurotransmitters and synaptic activity. Amyloid plaques in AD accumulate near dystrophic neurons and reactive astrocytes. B. Cordell, Annu. Rev. Pharmacol. Toxicol. 34, 69 (1994); D. J. Selkoe, Annu. Rev. Neurosci. 17, 489 (1994). The activation of neurotransmitter receptors, which are coupled to phosphotidylinositol (PI) hydrolysis or to protein kinase C (PKC) activation, can promote APP metabolism and decrease amyloid formation. R. M. Nitsch, B. E. Slack, R. J. Wurtman, J. H. Growdon, Science 258, 304 (1992); B. A. Wolf et al., J. Biol. Chem. 270, 4916 (1995); J. D. Buxbaum, A. A. Ruefli, C. A. Parker, A. M. Cypess, P. Greengard, Proc. Natl. Acad. Sci. U.S.A. 91, 4489 (1994); R. K. K. Lee, R. J. Wurtman, A. J. Cox, R. M. Nitsch, Ibid., 92, 8083 (1995); Ulus and Wurtman, J. Pharm..Exp.Ther., 281,149 (1997); Lee et al., PNAS USA, 92, 8083 (1995). Activation of neurotransmitters coupled to cAMP production suppresses both constitutive and PKC/PI-stimulated APPs secretion in astroglioma cells and in primary astrocytes. Eftimiopoulos et al., J. Neurochem., 67, 872 (1996); Lee et al., J. Neurochem., 68,1830 (1997). Cyclic AMP and PK activators also increase A production, Marambaud et al., Br. J. Pharmacol. 126, 1186 (1999). The drastic alterations in neurotransmitter levels and second messenger signalling created by neurodegeneration and synapse loss in AD may disrupt APP processing in ways that promote the accumulation of amyloidogenic or neurotoxic APP fragments. In contrast, the loss of various neurotransmitters in AD may increase cellular levels of APP holoprotein containing amyloidogenic or neurotoxic peptides due to a decrease in proper APP metabolism. B. A. Yankner et al., Science, 245, 417 (1989); M. R. Kozlowski, A. Spanoyannnis, S. P. Manly, S. A. Fidel, R. L. Neve, J. Neurosci. 12, 1679 (1992).
Increased APP production in Down""s syndrome/Trisomy 21 is associated with a high incidence of AD at an early age due to the extra copy of the APP gene. Overexpression of APP in cell cultures and in transgenic mice is also associated with neurodegeneration and with age-related cognitive deficits, suggesting that overexpression of APP could contribute to the neuropathology of AD. K. Maruyama, K. Terakado, M. Usami, K. Yoshikawa, Nature, 347, 566 (1990); K. K. Hsiao et al., Neuron 15, 1203-1218 (1995); P. M. Moran, L. S. Higgins, B. Cordell, P. C. Moser, Proc. Natl. Acad. Sci. U.S.A. 92, 5341 (1995).
Several APP isoforms, ranging in size from 695-770 amino acids, are derived by differential splicing of a primary transcript. Of the three major APP isoforms, APP695 is predominantly expressed in neurons; APP751 and APP770, which harbor an additional Kunitz-type protease inhibitor (KPI) insert at the N-terminus, are predominantly expressed in astrocytes and appear to be increased in AD brain. T. E. Golde, S. Estes, M. Usiak, L. H. Younkin, S. G. Younkin, Neuron 4, 253 (1990); R. L. Neve, E. A. Finch, L. R. Dawes, Ibid., 1, 669 (1990); J. P. Anderson et al., EMBO J. 8, 3627 (1989); C. Nordstedt et al., Proc. Natl. Acad. Sci. U.S.A. 88, 8910 (1991). The decreased amounts of APP695 in postmortem AD brains may be due to neuronal loss. The increase in KPI-containing APP isoforms in AD and in regions surrounding senile plaques raises the possibility that transcriptional activation of APP synthesis in astrocytes contributes to AD neuropathology.
Aging, neurodegeneration and synapse loss in AD are associated with astrocyte proliferation and an upregulation of KPI-containing APP isoforms. See, e.g., A. Brun, X. Liu, C. Erikson, Neurodegeneration 4, 171 (1995); R. Schechter, S. H. C. Yen, R. D. Terry, J. Neuropathol. Exp. Neurol. 40, 95 (1981); L. A. Hansen, D. N. Armstrong, R. D. Terry, Neurobiol. Aging 8, 1 (1987); K. Iverfeldt, S. I. Walaas, P. Greengard, Proc. Natl. Acad. Sci. U.S.A. 90, 4146 (1993).
McGeer, P. L. et al., in The Lancet, 335, 1037 (1990), present the results of a retrospective study that revealed an apparently low incidence of Alzheimer""s Disease in rheumatoid arthritis patients. These authors propose the possibility that anti-inflammatory therapy confers some protection against AD. While provocative, the authors"" proposal is based solely on circumstantial evidence. This fact is not lost on the authors, who note three alternative explanations for their observations, in addition to the possible protective role of anti-inflammatory therapy.
Andersen, K. et al., in Neurology (August 1995) 45:1441, describe the results of their retrospective study. This article, perhaps, illustrates the care that one should take in conducting studies xe2x80x9cin hindsightxe2x80x9d because of the danger of over-interpretation or over-manipulation of the data in an effort to enhance any perceived differences. To their credit, the authors tempered their conclusions, stating that their findings are xe2x80x9ccompatiblexe2x80x9d with a possible protecting effect of NSAIDs (non-steroidal anti-inflammatory drugs) on the risk of AD. The authors fairly point out that important issues remain, including whether the presence of complement leads to neurodegeneration or whether the activation of complement is brought about by the cell""s need to phagocytose damaged neurons, how long one has to be exposed to NSAIDs to obtain clinically detectable results, and the need for studies that are better designed. The article adds that no relationship between NSAIDs exposure and cognitive function is found.
In contrast, an earlier article by Rich, J. B. et al., which appeared in Neurology (January 1995) 45:51, reported on the results of their review of the records of 210 Alzheimer""s patients. These authors concluded that patients on NSAIDs performed better on certain tests, including Mini-Mental State Examination, Boston Naming Test, delayed Benton Visual Retention Test, among others, versus non-NSAID patients. However, no significant difference is found in an even greater number of other tests performed, including Block Design, Immediate Benton Visual Retention Test, Gollin Incomplete Figures Test, to name a few. Recognizing the inherent limitations of their study, the authors state that xe2x80x9c[m]ethodologic limitations inherent in retrospective studies such as this one preclude us from addressing the specificity of the protective effects of NSAIDs.xe2x80x9d Indeed, the patients examined are likely to be on several types of medication at once.
In fact, in an earlier study by Lindsay, J. and co-workers reported in Neurology (November 1994) 44:2073, it is found that those with arthritis had a significantly reduced risk of Alzheimer""s disease. It is also found that the use of NSAIDs gave rise statistically to a lower risk. However, it is suggested that the presence of arthritis itself is the determinant in lowering the apparent risk for developing Alzheimer""s disease and not the taking of NSAIDs.
One has to go back even earlier to a study by Rogers, J. et al., in Neurology (August 1993) 43:1609, to find a controlled 6-month investigation involving the administration of 100-150 mg indomethacin (an NSAID) or placebo to mild or moderately impaired Alzheimer""s disease patients. These authors report that, based on a battery of cognitive tests, the indomethacin treatment appeared to protect those patients receiving indomethacin from the degree of cognitive decline exhibited by patients receiving placebo. If anything, this article, or any that have followed this article, suggests that the administration of indomethacin reduces the onset of dementia in Alzheimer""s patients. Never has it been disclosed or suggested that the administration of indomethacin prevents the overproduction of APP.
Astrocytes upregulate expression of glial fibrillary acidic protein (GFAP) as they transform from a resting state into process-bearing reactive astrocytes during aging and in brain injury. Eddleston and Mucke, Neurosci., 54, 15 (1993). GFAP levels are elevated in brain tissue and cerebrospinal fluid in AD [Wallin et al., Dementia, 7, 267 (1996)], suggesting that reactive astrocytes may contribute to the neuropathology. Furthermore, persistent and rapid elevations in APP immunoreactivity have been observed in GFAP-positive astrocytes following brain injury. Siman et al., J.Neurosci., 3, 275 (1989); Banati and Kreutzberg, J. Cereb. Blood Flow Metab., 12, 257 (1995). In the AD brain , the loss of synapses is associated with an increase in the number of GFAP-positive astrocytes [Brun et al., Neurodegeneration, 4, 171 (1995)], and increases in KPI-containing APP mRNA in the frontal cortex have also been attributed to the astrocytic response during neuronal damage [Golde et al., Neuron, 4, 253 (1990)]. It seems that the loss of synapses and neurons in AD might initiate a pathological cascade that includes APP synthesis by reactive astrocytes.
Cytosolic phospholipase A2, which releases arachidonic acid from cellular phospholipids, is elevated in AD brain and after transient global ischemia. Stephenson et al., Neurobiol. Disease, 3, 51 (1996); Clemens et al., Stroke, 27, 527 (1996). The cyclooxygenation of arachidonic acid, catalyzed by two forms of cyclo-oxygenase (COX), COX-1 and COX-2, produces prostaglandins which, in turn, regulate neurotransmission, immune and inflammatory responses by activating receptors coupled to cAMP formation. Goetzl et al., FASEB J., 9, 1051 (1995). We have discovered that cAMP elevations caused by activation of neurotransmitter receptors increased APP mRNA and holoprotein production in astrocytes. Lee et al., PNAS USA, 94, 5422 (1997). As discussed herein, it is now shown that activation of prostaglandin E2 (PG E2) receptors coupled to increased cAMP formation also stimulates the synthesis of APP mRNA and holoprotein. This effect appears to be mediated by cAMP dependent protein kinases, and can be inhibited by various substances, including immunosuppressants, immunophilin ligands, and ion-channel modulators. Portions of this work have been presented as an abstract. Lee et al., J.Neurochem. (supp), 69, S103B (1997).
Epidemiologic and clinical data suggest that the use non-steroidal anti-inflammatory drugs (NSAIDs) delays the onset of AD and reduces the progression of pathologic symptoms in Alzheimer""s disease. McGeer and McGeer, Brain Res. Rev. 21, 195 (1995). Aspirin, like most NSAIDs, prevent inflammation and pain by inhibiting both COX-1 and COX-2 enzymes. Resveratrol, a phenolic antioxidant and COX inhibitor found in grapes, inhibits prostaglandin production, and has anti-cancer and anti-inflammatory properties. Jang et al., Science 275, 218 (1997).
Most traditional NSAIDs are more potent against the COX-1 form than against the COX-2 form of cyclo-oxygenase. COX-1 is constitutively expressed in most tissues; regulates the production of essential prostaglandins in kidney and gastric mucosa; and is essential for platelet thromboxane production. In contrast, COX-2 is rapidly inducible in response to cytokines, inflammation, and injury. The long-term use of non-specific COX inhibitors such as aspirin and related NSAIDs is potentially toxic to the kidney, and can cause ulcers and gastrointestinal bleeding. Allison et al., N. Engl. J. Med. 327, 749 (1992); Wallace Can. J. Physiol. Pharmacol. 72, 1493 (1994). COX-2 mRNA is elevated in the post-mortem brains of those with AD. Pasinetti and Aisen, Neuroscience, 87, 319-324.
Highly selective COX-2 inhibitors such as DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone) and DFP (5,5-dimethyl-3-isopropyloxy-4-(4xe2x80x2-methylsulfonylphenyl)-2(5H)-furanone) are orally bioactive drugs. Riendeau et al., Br. J. Pharmacol. 121, 105 (1997). These drugs prevent the conversion of arachidonic acid to prostaglandins.
In U.S. Pat. No. 5,385,915, Buxbaum et al. describe methods and compositions for affecting APP processing by the administration of agents that regulate protein phosphorylation, namely agents that regulate kinases or phosphatases. The modulation of APP processing leads, in turn, to the regulation of the production of xcex2/A4 peptide, a peptide that accumulates in amyloidogenic plaques. See, e.g., col.6, lines 8-10. Hence, Buxbaum et al. teach that one""s objective should focus on the search for agents that alter the metabolism of APP. They make no mention, teaching, or suggestion that the step preceding the processing of APP, that is, the expression, production, or formation of APP, itself, can be at all affected by select groups of substances. Indeed, as Buxbaum et al. state (at col. 21, lines 7-9), xe2x80x9cthe effects observed are attributable to changes in APP metabolism rather than APP transcriptionxe2x80x9d (emphasis added). Consistent with this notion, the claims of Buxbaum et al. are drawn to a method of regulating phosphorylation of proteins that control the processing of APP.
Similarly, in U.S. Pat. No. 5,242,932, Gandy et al. disclose and claim a method of modulating or affecting the intracellular trafficking and processing of APP in the mammalian cell.
For additional background information on the processing of APP, release of APP derivatives, or the processing, degradation and secretion of xcex2/A4 APP, the interested reader is referred to the following publications: Nitsch, R. M. et al. Science (1992) 258:304; Lee, R. K. K. et al. Proc. Nat""l. Acad. Sci. USA (1995) 92:8083; Caporaso, G. L. et al. Proc. Nat""l. Acad. Sci. USA (1992) 89:3055; Caporaso, G. L. et al. Proc. Nat""l. Acad. Sci. USA (1992) 89:2252; and Buxbaum, J. D. et al. Proc. Nat""l. Acad. Sci. USA (1992) 89:10075.
Accordingly, it is an object of the present invention to provide methods and compositions that modulate or regulate the production or formation of APP in patients, including the expression of APP gene products and the transcription or translation of the APP gene in brain cells. For example, the production of APP by mammalian cells, in particular, by cells in the brain, can be increased or reduced.
In attaining this objective, it is also an objective of the invention to inhibit excessive amyloid formation, prevent neurite dystrophy and alleviate pathological symptoms, such as neurodegeneration or cognitive deficits that may arise from the negative effects of inappropriately expressed, produced, or formed amounts of APP.
Furthermore, the present invention seeks to provide compositions and methods that alleviate the detrimental effects of inappropriate APP production arising from overstimulation of receptors, particularly those coupled to cAMP formation.
It is also an object of the invention to provide relief from the debilitating effects of injury or trauma to the brain, as well as neurological diseases and neurodegenerative disorders, such as Alzheimer""s, Parkinson""s, or Lou Gehrig""s Disease (amyotrophic lateral sclerosis), multiple sclerosis and the like, which may have their roots in the formation or presence of amyloid plaques.
It has now been discovered that prolonged activation of receptors that are coupled to increased cAMP formation in cortical astrocytes upregulates both APP mRNA levels and APP holoprotein bearing the KPI insert. It has further been discovered that such overproduction, which it is shown can result from the body""s response to brain injury or trauma, can be inhibited by certain substances, including agents that inhibit an inflammatory response and immune system suppressants or immunophilin ligands.
In particular, it has been discovered that the immune or inflammatory response of astrocytes to brain injury accelerates the formation of amyloid plaques. It has also been shown that cAMP signaling regulates the astrocytic response to neuronal injury, while also stimulating transcriptional activation of the APP gene. Further, it is shown that prostaglandins, which are coupled to cAMP, also stimulate APP overexpression.
What is more, it has been shown that certain substances, including immune system suppressants (agents that inhibit an inflammatory response, and the like) or immunophilin ligands inhibit this abnormal increase in APP synthesis (mRNA and cell-associated protein), which is caused by elevations in cAMP levels. Thus, these substances can be used to prevent APP over-expression in brain cells.
It has further been discovered that ion-gated channels can regulate APP overexpression. This is based on the observations, discussed herein, that the activation of nicotinic receptors in cultured astrocytes increases cellular levels of APP, as well as the observation that ionchannel modulators, such as calcium channel blockers and calcium/calmodulin kinase inhibitors, partially inhibit the stimulatory effect of prostaglandins on APP synthesis.
It has further been discovered that non-steroidal antiinflammatory agents (NSAIDS), such as inhibitors of cyclo-oxygenase activity including, but not limited to, aspirin also prevent APP overexpression and the overproduction of amyloidogenic peptides.
It has still further been discovered that non-steroidal antiinflammatory agents (NSAIDS), such as specific inhibitors of cyclo-oxygenase type 2 activity including, but not limited to, DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4 methylsulfonyl)phenyl-2(5H)-furanone), DFP (5,5-dimethyl-3-isopropyloxy-4-(4xe2x80x2-methylsulfonylphenyl)-2(5H)-furanone), and resveratrol, can also prevent APP overexpression and the overproduction of amyloidogenic peptides.
Furthermore, it has been discovered that NSAID cyclooxygenase type 2 inhibitors also increase secretion of soluble APP, and reduce intracellular APP holoprotein.
The present invention also contemplates and provides an assay for identifying or screening potential drugs that can inhibit the excessive or inappropriate production of amyloidogenic or neurotoxic APP fragments. For example, such an assay may utilize a tissue or cell culture comprising brain cells, as described herein.
The invention also contemplates a method by which the overexpression of APP is deliberately effected, followed by the promotion or stimulation of APP metabolism to provide soluble APP (APPs). The former step can be attained by, for example, cAMP signaling, while the latter process can be accomplished, for example, by the activation of protein kinase C (PKC) or of neurotransmitter agonists (e.g., via m1, m3, serotoninergic, or metabotropic glutamate receptors) which increase phophotidylinositol (PI) hydrolysis. Consequently, increased amounts of APPs are secreted into the medium, and the formation of amyloidogenic Axcex2 peptides is disrupted. It is believed that secreted APPs have neurotrophic and neuroprotective functions. Secreted APPs have been shown to promote neurite outgrowth and maintain synapse. It is believed further that increased APPs secretion promotes synaptic transmission and neuronal regeneration (e.g., via neurite or axonal outgrowth). The net result is the conversion of an amyloidogenic event (i.e., APP overexpression and Axcex2 formation) into a neurotrophic event (i.e., APPs secretion).
The invention further contemplates that decreasing overexpression of amyloidogenic APP and amyloidogenic APP peptides is beneficial in treatment of subjects with neurodegenerative conditions, including immune or inflammatory conditions associated with Alzheimers Disease, stroke, cerebral ischemia, de-myelinating conditions, and mechanical injury. Decreasing overexpression of amyloidogenic APP and amyloidogenic APP peptides is also contemplated in the present invention as beneficial in the prevention of neurodegenerative diseases including Alzheimers Disease.
These and other objects of the invention will be evident to those of ordinary skill from a consideration of the discussions and descriptions provided in this specification, including the detailed description of the preferred embodiments.